Identification of sites on epidermal growth factor receptors which are phosphorylated by pp60src in vitro

摘要

The Epidermal Growth Factor Receptor (EGF-R) becomes constitutively tyrosine phosphorylated on two novel sites in v-Src trans formed cells, and these phosphurylations arc associated with enhanced signaling activity [)], To determine whether Src could directly phosphorylate these sites, we have examined the ability of the Src kinase to phosphoryiatc both wild-type and kinase-defeetive EGF-Rs in vitro. Although purified Src could phosphorylate EGF-Rs, the pattern of phosphorylation sites was not identical to what was previously found in vivo [1]: Src in vitro directly phosphorylated EGF-Rs on one autophosphorylation site (Tyr 1173) which was not a site of Src-induced in vivo phosphorylation, suggesting the in vivo inaccessibility of this site. One Src-speeific in vitro phosphorylation .site (Tyr 703) appeared to correspond to one of the in vivo Src-induced sites (sPY2), but the other Src-speeific in vivo site (sPYl) was not significantly phosphorylated in vitro, raising the possibility of a Src-induced tyrosine kinase cascade. The ability of Src to phosphorylate the EGF-R is consistent with the suggestion that the receptor can function as a kinase substrate independent of its intrinsic enzymatic activity, as implied by recent studies on signaling by kinase-defeetive EGF-Rs.